The goal of this R21 grant application is to develop combinations of novel synthetic immunostimulatory agents that maximize host resistance to infectious agents. Many pathogens important for biodefense are first encountered at mucosal or epithelial sites in the respiratory tree or the gastrointestinal tract. The initial host defense against these agents, as well as the development of a protective immune response, requires the prompt activation of the innate immune system at the portal of entry. Dendritic cells (DCs) at mucosal sites play a crucial role in immune responses against infections by sensing microbial invasion through their Toll-like receptors (TLRs). However, innate immune system activation by microbial agents may be compromised at the mucosal surfaces of normal people for two reasons. First, the antigens encountered by the musocal immune system often do not induce vigorous TH1 and cytotoxic responses, because of a tolerogenic local environment. Second, many viruses and bacteria express decoy or inhibitory proteins that impair efficient TLR signaling. Thus, there is a strong rationale for the development of drugs that can overcome these two barriers to innate immune system activation. Based upon preliminary experiments conducted in our own laboratory, we believe that barriers to innate immune system activation can be bypassed by delivering to mucosal surfaces a combination of TLR ligands (TLR-L), which synergistically activate DCs. Our experiments will focus on newly developed synthetic activators of TLR7 and TLR9, because these TLRs are co-expressed in the endosomal compartments of DCs. In some cases, the TLR ligands will be combined with either a macrophage-targeted activator of TLR8, and/or with synthetic inhibitors of inosine monophosphate dehydrogenase (IMPDH), which have been shown in preliminary experiments to potentiate TLR signaling by inducing interferon regulatory factors 3 and 7 (IRF-3, IRF-7).